We developed an Engineered Early Embryonic Cardiac Tissue, termed EEECT, using embryonic rat cardiac cells isolated during the period of cardiac morphogenesis, in order to investigate the regulation of immature cardiomyocyte (CM) proliferation and differentiation and to generate implantable tissues with optimal properties for cardiac repair. Our EEECT construct preserves the unique proliferative and contractile properties of immature CM, including a proliferative response to cyclic mechanical stretch. With prolonged culture EEECT acquires a post-natal phenotype (reduced proliferation and increased force production). Preliminary data show that EEECT implanted onto injured adult rat LV survive and improve LV diastolic and systolic function. In this revised R01-A2 proposal we identify molecular pathways that regulate EEECT CM proliferation and test our paradigm that EEECT is an optimal CM-rich tissue construct for cardiac repair. Specific Aim 1: Define molecular pathways that regulate EEECT CM proliferation during the transition from fetal to post-natal CM phenotype. We hypothesize that the regulation of CM proliferation and growth involves 2 concurrent processes: (1) CM proliferation to expand myocardial mass via p38 mitogen-activatedprotein kinase (p38MAPK) and Akt;and (2) CM sarcomere maturation and growth of non-proliferating CM via integrin-linked kinase (ILK), p38MAPK, and Akt. We will use our in vitro EEECT model (generated from ED 14 embryonic rat cardiac cells) to define the impact of (1) cyclic mechanical loading;(2) stimulation or blockade of ILK, p38MAPK, and Akt;and (3) thyroid hormone treatment on CM proliferation and the phenotypic transition. Post-treatment characterization of EEECT will include: (1) histologic measures of CM proliferation, differentiation, and apoptosis;(2) biomechanical properties;(3) protein content and kinase activities;and (4) genome-wide changes in RNA transcript expression patterns. Specific Aim 2: Determine the fate of EEECT following implantation onto injured adult myocardium and the contribution of EEECT to recipient myocardial functional recovery and remodeling. We hypothesize implanted EEECT (1) display sustained CM proliferation and limited cell death;(2) positively contribute to the diastolic and systolic functional recovery of infarcted myocardium;and (3) become vascularized. We implant GFP+ transgenic rat EEECTs onto syngenic rat LV 2 weeks following myocardial infarction. Outcome assays at 3 days, 2 weeks, and 8 weeks include LV functional assay and histologic characterization of cell proliferation, cell phenotype, cell-cell coupling, and vascularization. We further hypothesize that in vitro treatments that increase EEECT CM proliferation will increase in vivo EEECT CM survival and functional recovery. PUBLIC HEALTH RELEVANCE: Our proposed experiments first identify pathways that regulate cardiomyocyte proliferation and maturation within an Engineered Early Embryonic Cardiac Tissue (EEECT) generated using tissue culture and immature cardiac cells. We then evaluate the success of EEECT implantation onto the surface of the injured adult heart (myocardial infarction) as part of a cardiac repair and regeneration strategy.